The present invention relates to compounds which repress DNA transcription. Specifically, the invention relates to a thirteen amino acid polypeptide sequence which is able to autonomously function as a transcription repressor domain through its ability to independently bind mSin3A. The invention further relates to chimeric transcriptional repressors which comprise the thirteen amino acid polypeptide and a DNA-binding molecule.
Precise changes in gene expression are crucial to both normal and disease processes, Gene expression is regulated by DNA-binding transcription factors and the proteins that interact with these DNA-bound factors. Coactivators and corepressors mediate the ability of DNA-bound transcription factors to modulate gene expressionxe2x80x94coactivators by increasing the expression of genes, and corepressors by down-regulating the expression of genes.
Transcriptional regulation depends on the assembly of large multiprotein complexes. For example, the preinitiation complex (Kadonaga, J. T. Cell 92: 307-313 (1998)), chromatin remodeling complexes (Cairns, B. R. Trends Biochem. Sci. 23:20-25 (1998), Wu, C. J Biol. Chem. 272:28171-28174 (1997)), and histone deacetylase-containing corepressor complexes (Rundlett et al., Proc. Natl. Acad. Sci. USA 93:14503-14508 (1996), Zhang et al., Cell 95:279-289 (1998)) have been shown to be in the 1-2xc3x97106 dalton size range. Molecular connections between proteins in these molecular machines, and the structural basis of their assembly, are not well understood. Initially, transcription repression domains were defined by structure/function analysis, which revealed that, like activation domains, they are more likely to contain particular amino acids rather than have easily identifiable protein-protein interaction domains. This finding led to the hypothesis that activation and repression domains share similar molecular targets and that the structure of the activation or repression domain in itself was not required for function. Transciptional repressors function by at least three distinct mechanisms: by direct contact with components of the basal transcriptional machinery, e.g. even-skipped (Um et al., Mol. Cell. Biol. 15:5007-5016 (1995)), Dr1 (Yeung et al., Genes Dev. 8424:2097-2109 (1994)), and MOT1 (Auble et al., Genes Dev. 8:1920-1934 (1994)); by tethering histone deacetylase-containing corepressor complexes to the promoter, e.g. the Mad family (Hassig et al., Cell 89:341-347 (1997), Laherty et al., Cell 89:349-356 (1997)), Rb (Brehm et al., Nature 391:597-601 (1998), Magnaghi-Jaulin et al., Nature 391:601-605 (1998), and Luo et al., Cell 92:463-473 (1998)), and MeCP2 (Jones et al., Nat. Genet. 19:187-191 (1998), Nan et al., Nature 393:386-389 (1998)); or by tethering corepressors that lack deacetylase activity to the promoter, e.g. hairy (Paroush et al., Cell 79:805-815 (1994)) and MATxcex12-MCM1 (Kadosh and Struhl, Cell 89:365-371 (1997)). In each of these cases, little or no structural data are available for the repression domain. In contrast, one theme that has emerged recently from the study of activation domains is that relatively short stretches of amino acids can adopt amphipathic xcex1-helical structures and mediate stable functional interactions between transcriptional activators and coactivators. Kussie et al., Science 274:948-953 (1996), Radhakrishnan, et al., Cell 91:741-752 (1997), and Uesugi et al., Science 277:1310-1313 (1997).
Reversible acetylation of the amino-terminal tails of core histones plays an important role in the regulation of gene expression. In general, regions of chromatin that are hyper-acetylated are transcriptionally active, while hypoacetylated regions are silenced. Grunstein, M., Nature 389-352 (1997). The recent discovery that several transcriptional co-activators are histone acetyltransferases and that co-repressor complexes contain histone deacetylases as active components has provided a mechanistic basis for this correlation. Wolffe and Pruss, Cell 84:817-819 (1996), Hassig et al., Curr. Opin. Chem. Biol. 1:300-308 (1997), Grant et al., Trends Cell Biol. 8:193-197 (1998), Struhl, Genes Dev. 12:599-606 (1998), Davie, Curr. Opin. Genet. Dev. 8:173-178 (1998). mSin3A and mSin3B were identified as corepressors required for the transciptional and biological activities of the Mad proteins. Ayer et al., Cell 80:767-776 (1995); Schreiber-Angus et al., Cell 80:777-786 (1995). mSin3A has recently been shown to be a component of a large multi-protein complex(s) that also contains the histone deacetylases HDAC1 and HDAC2 in apparently stoichiometric amounts. The enzymatic activities of the mSin3A-bound HDACs are required for full transcriptional repression by the Mad family proteins. Hassig et al., Cell 89:341-347 (1997); Laherty et al., Cell 89:349-356 (1997), Zhang et al., Cell 89:357-364 (1997). Subsequently, the mSin3A-HDAC complex has been implicated as a corepressor utilized by a diverse and rapidly expanding collection of transcriptional repressors, including RXR, MeCP2, estrogen receptor, RPX, and Pit1. (Jones et al., Nat. Genet. 19:187-191 (1998), Struhl, Genes Dev. 12:599-606 (1998), Laherty et al., Mol. Cell 2:33-42 (1998), Heinzel et al., Nature 387:43-48 (1997), Nagy et al., Cell 89:373-380 (1997).
mSin3A and mSin3B and their Saccharomyces cerevisiae orthologue SIN3 each contain four similar domains each suggested to form two amphipathic xcex1-helices separated by a flexible linker. Ayer et al., Cell 80:767-776 (1995), Wang et al., Mol. Cell Biol. 10:5927-5936 (1990). These regions, termed PAH domains for paired amphipathic xcex1-helix, were originally proposed to function as protein-protein interaction domains. Wang et al., Mol. Cell Biol. 10:5927-5936 (1990). Recent experiments have demonstrated this to be the case. For example, Mad proteins interact with PAH2 (Ayer et al., Cell 80:767-776 (1995), Schreiber-Angus et al., Cell 80:777-786 (1995)), a repression domain of the nuclear hormone corepressor N-CoR interacts with PAH1 (Heinzel et al., Nature 387:43-48 (1997), Alland et al., Nature 387:49-55 (1997)), and the mSin3 interacting protein SAP30 binds to PAH3. (Laherty et al., Mol. Cell 2:33-42 (1998). The four PAH domains of the different Sin3 proteins are highly conserved. For example, PAH2 is 90% similar between mSin3A and mSin3B and it is approximately 70% similar to the PAH2 domain of S. cerevisiae SIN3 (Ayer et al., Cell 80:767-776 (1995)) and recently identified SIN3 homologues from Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana. Within a given protein, the four PAH domains are roughly 45% similar with the hydrophobic positions of the putative amphipathic xcex1-helices being most highly conserved, suggesting that PAH domains may share structural features. Ayer et al., Cell 80:767-776 (1995); see also Kasten et al., Mol. Cell. Biol. 16:4215-4221 (1996) (demonstrating that human Mad1 can interact with yeast SIN3). With the exception of the Mad family, the domains required for Sin3 binding of the other SIN3 interacting proteins, SAP30, SAP18, N-CoR, UME6, HDAC1, and HDAC2, etc., share no obvious sequence similarity.
The Mad family of basic region-helix-loop-helix-leucine zipper (bHLHZip) proteins functions as transcriptional repressors and antagonize the transcriptional and transforning activity of the Myc proto-oncogenes. Ayer et al., Cell 72:211-222 (1993), Koskinen et al., Cell Growth Differ. 6:623-629 (1995), Lahoz et al., Proc. Natl. Acad. Sci. USA 91:5503-5507 (1994), Hurlin et al., EMBO J. 14:5646-5659 (1995), and Vastrik et al., J. Cell Biol. 128:1197-1208 (1995). Currently, four Mad family members have been identified: Mad1, Mxi1, Mad3, and Mad4. Ayer et al., Cell 72:211-222 (1993), Hurlin et al., EMBO J. 14:5646-5659 (1995), Zervos et al., Cell 72:223-232 (1993). These proteins share extensive sequence homology throughout their entire open reading frames, with the highest degree of conservation within the bHLHZip and for mSin3 interaction domains (SID). Hurlin et al., EMBO J. 14:5646-5659 (1995). The bHLHZip domain is required for dimerization with the bHLHZip protein Max and DNA binding, while the SID is required for interaction with mSIN3A or mSin3B. Schreiber-Angus et al., Cell 80:777-786 (1995), Ayer et al., Cell 72:211-222 (1993), and Hurlin et al., EMBO J. 14:5646-5659 (1995). This SID sequence from Mad1 has been modeled as an amphipathic xcex1-helix. Ayer et al., Cell 80:767-776 (1995). Recently, another bHLHZip protein termed Mnt, which shares homology to the Mad family within these two regions, has been identified. Mnt also interacts with Max and can repress transcription in a mSin3-dependent manner and therefore appears to be functionally equivalent to the Mad family proteins. Hurlin et al., Genes Dev. 11:44-58 (1997).
Several lines of experimental evidence suggest that interaction between the Mad proteins and Mnt and mSin3A or mSin3B is critical for their function as transcriptional repressors. Mad1 proteins with point mutations in the SID no longer repress transcription, block Myc+Ras cotransformation, or arrest cells in the G1 phase of the cell cycle. Ayer et al., Cell 80:767-776 (1995), Koskinen et al., Cell Growth Differ. 6:623-629 (1995), Roussel et al., Mol. Cell Biol. 16:2796-2801 (1996). Similarly, deletions of amino-terminal regions that contain the SID in Mad3, Mad4, and Mnt severely affect their biological function. Hurlin et al., EMBO J. 14:5646-5659 (1995), Hurlin et al., Genes Dev. 11:44-58 (1997). Finally, Mxi1 is encoded by two alternatively spliced mRNAs, only one of which encodes a Mxi1 protein with a SID. This protein, Mxi1-SR, is much more potent at blocking Myc+Ras cotransformation than is an Mxi1 isoform which lacks a SID. Schreiber-Angus et al., Cell 80:777-786 (1995).
Since current methods for modulating gene expression are laborious and often ineffective, the development of small molecule repressors would constitute a major advance in the art. Additionally, since many human diseases are characterized by the misexpression of a gene, the development of new methods for safely and effectively repressing the transcription of such genes would also be an advance in the art.
From the foregoing, it will be appreciated that it would be an advancement in the art to provide a peptide comprising the minimal SID domain necessary to interact with the PAH2 region of mSin3A/B. It would be a further advancement to provide chimeric transcriptional repressors that could repress the expression of selected genes. It would also be an advancement to provide methods that allow selective repression of gene expression.
Such compounds and methods are disclosed herein.
The present invention relates to a small polypeptide and variants thereof that are capable of interacting with the Sin3 corepressor. These polypeptides are portable repression domains that confer the property of dominant transcriptional repression upon heterologous DNA binding molecules. The polypeptides of the invention permit the synthesis of small molecule regulators of gene expression. Further, since such a polypeptide constitutes a minimal repression domain, it greatly facilitates the production of chimeric transcriptional regulators by conventional gene cloning techniques. The present invention therefore provides chimeric transcriptional repressors comprising such polypeptides and a DNA-binding domain. In certain embodiments, the DNA-binding domain is a polyamide. Preferably, the polyamide is capable of binding a regulatory region of a gene. In certain other embodiments, the DNA-binding domain comprises a zinc finger domain. Preferably, the zinc finger domain is capable of recognizing a regulatory region of a gene.
Other DNA binding domains are known in the art and may be used to construct chimeric transcriptional regulators of the present invention. Known DNA binding domains include BHLH (basic helix-loop-helix), BHLHLZ (basic helix-loop-helix leucine zipper), BZIP (basic zipper), homeodomains, POU domains, and ETS domains. Additionally, a number of proteins that bind DNA (e.g., the p53 protein) are known that do not contain classical DNA binding motifs. One of skill in the art would appreciate that the DNA binding motifs of such proteins could be used to construct chimeric transcriptional regulators of the present invention.
Chimeric transcriptional regulators of the present invention may include one or more than one SID. In certain embodiments, a chimeric transcriptional regulator of the present invention comprises multiple repeated SIDs.
The present invention also provides derivatives of the polypeptide having both higher and lower Sin3 binding affinities than the wild-type. In certain embodiments, these derivatives are at least 77% identical to the amino acid sequence of SEQ ID NO: 1xe2x80x94that is, at least ten out of thirteen residues are identical. In certain other embodiments, the amino acid sequences of these derivatives are at least 85% or at least 92% identical to the amino acid sequence of SEQ ID NO: 1.
In certain embodiments, the SID is less than thirty-five amino acids in length. In certain preferred embodiments, the SID is about twenty or fewer amino acids in length. In other embodiments, the SID is fourteen or fifteen amino acids in length. In certain preferred embodiments, the SID is thirteen amino acids in length.
The present invention also provides methods for creating transcriptional regulators. In certain embodiments, such methods comprise the steps of (1) synthesizing an mSin3A-binding molecule and (2) linking the mSin3A-binding molecule to a heterologous DNA-binding molecule. In certain embodiments, the mSin3A-binding molecule is at least 77%, at least 85%, or at least 92% identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the DNA-binding molecule comprises a polyamide capable of recognizing a regulatory DNA sequence. In certain other embodiments, the DNA-binding molecule comprises a zinc finger domain capable of recognizing a regulatory DNA sequence.
The mSin3A-binding molecule and the DNA-binding molecule may be linked covalently or non-covalently. In certain embodiments, the linkage is covalent and the linking is accomplished using recombinant DNA technology. In certain other embodiments, the linkage is covalent and the linking is accomplished using the standard protection and coupling chemistry used in the art for BOC peptide synthesis.
In certain other embodiments, the mSin3A-binding molecule and the DNA-binding molecule are linked non-covalently and the linkage is accomplished using biotin-streptavidin interactions. In certain other embodiments, conditional dimerization technology is used to achieve a non-covalent linkage. In yet other embodiments, non-covalent linkage is accomplished using FK506 and FKBP interactions. For example, in one embodiment of the present invention, FK506 binding domains are attached to both the mSin3A-binding molecule and to the DNA-binding molecule. These two molecules will remain apart in the absence of a dimerizer. Upon addition of a dimerizer, such as the synthetic ligand FK1012, the two molecules would fuse.
The present invention also comprises methods of using these polypeptides to repress transcription of a selected gene in a cell. In certain embodiments of the present invention, the selected gene comprises at least one regulatory region and the methods comprise the steps of (1) generating a fusion comprising a DNA-binding molecule and a SID, wherein the DNA-binding molecule binds to a target nucleotide sequence within the at least one regulatory region and the SID comprises an amino acid sequence that is at least 77% identical to the amino acid sequence of SEQ ID NO: 1; and (2) introducing the fusion into the cell under conditions such that the DNA-binding molecule binds to a target nucleotide sequence and transcription is repressed. In certain other embodiments, the DNA-binding molecule binds to a sequence within a structural feature of the gene, such as an intron or exon.
The term xe2x80x9crepressionxe2x80x9d refers to a decreased level of transcription when compared to the level of transcription of the same gene in a comparable cell without the fusion. Techniques for conducting such a comparison are well-known in the art. In certain embodiments, the level of repression is 90%xe2x80x94that is, the transcription of the selected gene is decreased by 90% when compared to the transcription of the same gene in a comparable cell into which the fusion has not been introduced. In certain other embodiments, the level of repression is less than 5%, 5%, 10%, 25%, 50%, 75%, 95%, 99%, or greater than 99%.
The present invention also provides methods for creating a functional disruption of a selected gene in a cell, wherein the selected gene comprises at least one regulatory region and the methods comprise the steps of (1) determrining the sequence of the at least one regulatory region of the gene; (2) designing a DNA-binding peptide which binds a sequence within the regulatory region; (3) constructing a nucleic acid molecule comprising nucleotides which code for a chimeric transcriptional repressor, wherein said chimeric transcriptional repressor comprises the DNA-binding molecule and a SID that comprises an amino acid sequence that is at least 77% identical to the amino acid sequence of SEQ ID NO: 1; and (4) inserting the nucleic acid molecule into the genome of the cell. In certain preferred embodiments, the nucleic acid molecule further comprises an inducible promoter.
The present invention further provides methods for correcting a disease characterized by misexpression of a gene. In certain embodiments, the selected gene comprises at least one regulatory region and the methods comprise the steps of (1) determining the sequence of the at least one regulatory region of the gene; (2) designing a DNA-binding peptide which binds a sequence within the regulatory region; (3) constructing a nucleic acid molecule comprising nucleotides which code for a chimeric transcriptional repressor, wherein said chimeric transcriptional repressor comprises the DNA-binding molecule and a SID comprising an amino acid sequence that is at least 77% identical to the amino acid sequence of SEQ ID NO: 1; and (4) inserting the nucleic acid molecule into the genome of the cell.
These and other features of the present invention will become apparent upon reference to the accompanying figures and upon reading the following detailed description and appended claims.